The purpose of these studies is to develop imaging techniques to monitor sub-cellular structures and processes, in vivo. We have been systematically developing an in vivo optical microscopy system that is adapted to biological tissues and structures rather than forcing an animal on a conventional microscope stage. The following major findings were made over the last year: 1) The tight coupling of mitochondria across muscle cells in the mitochondria reticulum is a risk to the cell. If one mitochondria fails, it could pull down the entire mitochondrial network just like a short circuit in a house. We have recently completed a study that demonstrates a rapid fail safe system is in place that removed damaged mitochondria from the network. Our current hypothesis is that this fail safe, or circuit breaker, mechanism is structural in nature representing the physical uncoupling of the mitochondria from the network and cytoskeleton. This is based on the new hypothesis that the mitochondrial reticulum is under tension, that is stretched by the cytoskeleton to hold these complex positions, until damage is detected and the mitochondria is released and springs back to its native spherical structure. This process has been directly observed in muscle cells during local disruption of mitochondrial function. This adds an entirely new regulatory aspect to the mitochondria dealing with it distribution and structure to meet cellular needs. 2) Using our ability to monitor subcellular events rapidly in the living animal, we have completed a collaboration with Dr. Sinnis at Johns Hopkins to monitor the trafficking of malaria parasites upon inject via a simulated mosquito proboscis. The simulated proboscis is a specially designed fluorescent glass pipet that we can monitor in the animal with a 2-Photon excitation microscope. Using genetically labeled parasites with green fluorescent protein and mice ( with a vessel wall fluorescent protein) we have been able to observe the first few seconds of the inoculation process and observe how the parasites behave under the skin. We hope these studies will reveal how the parasites find and penetrate blood vessels to initiate the malaria infection. Most surprising in this study was the rather linear path that the parasites took in the tissue and that very few infections of the vasculature occur per injection suggesting that the homing of the parasites to the vascular is imperfect and can be potentially disrupted further. These data may provide a new strategy in preventing the malaria infection immediately after the inoculation.